Gaussian band analysis of absorption, fluorescence and photobleaching difference spectra of D1/D2/cytb-559 complex

A study of the absorption and fluorescence characteristics of the D1/D2/cytb-559 reaction centre complex of Photosystem II has been carried out by gaussian decomposition of absorption spectra both at room temperature and 72 K and of the room temperature fluorescence spectrum. A five component fit was found in which the absorption and fluorescence sub-bands could be connected by the Stepanov relation. The photobleaching and light-activated degradation in the dark of long wavelength pigments permitted a further characterisation of the absorption bands. The absorption (fluorescence) maxima of the five bands at room temperature are 660 nm (670 nm), 669 nm (675 nm), 675 nm (681 nm), 680 nm (683 nm), 681 nm (689 nm). A novel feature of this analysis is the presence of two approximately isoenergetic absorption bands near 680 nm at room temperature. The narrower one (FWHM=12.5 nm) is attributed to pheophytin while the broader band (FWHM=23 nm) is thought to be P680. The P680 band width is discussed in terms of homogeneous and site inhomogeous band broadening. The P680 fluorescence has a large Stokes shift (≈9 nm) and most fluorescence in the 690-700 nm range is associated with this chromophore.The three accessory pigment bands are broad (FWHM=17-24 nm) and the 660 nm gaussian is largely temperature insensitive thus indicating significant site inhomogeneous broadening.The very slight narrowing of the D1/D2/cytb-559 Qy absorption at crytogenic temperatures is discussed in terms of the coarse spectral inhomogeneity associated with the spectral forms and the apparently large site inhomogeneous broadening of short wavelength accessory pigments.